Structural Biology of Sterol Sensing and Transport[unreadable] [unreadable] Cholesterol homeostasis is fundamental to eukaryotic cell function and to human health. Cholesterol is regulated at the level of its uptake, synthesis, storage, transport, and metabolism. Cholesterol is transported between cell membranes by vesicular and non-vesicular mechanisms. The non-vesicular transport mechanisms involve the binding of cholesterol to soluble carrier proteins (or soluble domains of membrane proteins), which move cholesterol across aqueous gaps between membranes. Cholesterol and other sterol levels are monitored by regulatory proteins that contain specific binding sites for sterols. These include the transmembrane sterol sensing domain (SSD) proteins and Insig proteins, and the soluble START (StAR related) domain-containing protein and ORP (Oxysterol binding protein related protein) families. In some cases, the status of sterol binding proteins as transporters versus sensors remains ambiguous. The underlying principles of molecular recognition events are the same for both cases. This project seeks to understand molecular recognition in sterol sensing and transport using structural biology approaches. The goals of the project are to 1) determine structures of sterol binding proteins in complex with their physiological ligands; 2) determine the dynamics of sterol binding and dissociation by characterizing apo structures and using molecular dynamics to analyze the pathways connecting bound and apo states; and 3) relate structural features to biological function using mutational analysis of in vitro properties and in vivo functions.[unreadable] [unreadable] The oxysterol binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to man and are implicated in regulation of sterol pathways and in signal transduction. OSBP was first discovered as a cytosolic receptor for oxysterols that downregulate cholesterol synthesis. The cloning of OSBP led to the identification of a large family of OSBP-related proteins, the ORPs, with 7 members in S. cerevisiase and 12 in H. sapiens. ORPs are essential for life in eukaryotes. The deletion of all 7 ORPs leads to severe defects in sterol and lipid distribution and endocytosis in yeast, and OSBP is essential for embryonic development in mice. All ORPs contain a core OSBP-related domain (ORD), and many also contain pleckstrin homology (PH) domains, transmembrane regions, endoplasmic reticulum (ER)-targeting FFAT motifs, GOLD domains, and/or ankyrin repeats. These additional domains localize ORPs by binding to phosphoinositides, the ER protein VAP, and other targeting signals. The localization of ORPs is dynamic. Oxysterol binding changes the subcellular localization of certain ORPs from the cytosol to the Golgi or ER. ORPs can bind lipids other than oxysterols, including phosphoinositides and phosphatidic acid. OSBP is a cholesterol sensing regulator of two protein phosphatases, a PTPPBS family member, and Ser/Thr phosphatase PP2A. In previous work, we determined the structure of the full-length yeast ORP Osh4 at 1.5-1.9 resolution in complexes with ergosterol, cholesterol, and 7-, 20-, and 25-hydroxycholesterol. A single sterol molecule binds in a hydrophobic tunnel in a manner consistent with a transport function for ORPs. The entrance is blocked by a flexible N-terminal lid and surrounded by functionally critical basic residues. The structure of the open state of a lid-truncated form of Osh4 was determined at 2.5 resolution. Structural analysis and limited proteolysis show that sterol binding closes the lid and stabilizes a conformation favoring transport across aqueous barriers and transmitting signals. The unliganded structure exposes potential phospholipid-binding sites that are positioned for membrane docking and sterol exchange. Based on these observations we proposed a model in which sterol and membrane binding promote reciprocal conformational changes that facilitate a sterol transfer and signaling cycle.[unreadable] [unreadable] In the bound state, a 29 residue N-terminal lid region covers the opening of the cholesterol-binding tunnel of Osh4, preventing cholesterol exchange. Equilibrium and steered molecular dynamics (MD) simulations of Osh4 were carried out to characterize the mechanism of cholesterol exchange. While most of the structural core was stable during the simulations, the lid was partly opened in the apo equilibrium MD simulation. Helix alpha 7, which undergoes the largest conformational change in the crystallized bound and apo states, is conformationally coupled to the opening of the lid. The movement of alpha 7 helps create a docking site for donor or acceptor membranes in the open state. In the steered MD simulations of cholesterol dissociation, we observed complete opening of the lid covering the cholesterol-binding tunnel. Cholesterol was found to exit the binding pocket in a step-wise process involving (i) the breaking of water-mediated hydrogen bonds and van der Waals contacts within the binding pocket, (ii) opening of the lid covering the binding pocket, and (iii) breakage of transient cholesterol contacts with the rim of the pocket and hydrophobic residues on the interior face of the lid.